The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and HDL is responsible for cholesterol disposal from the body via reverse cholesterol transport (RCT) and is critical in the prevention of dysfunctional HDL formation and atherosclerosis. The long-term objective of our research is to understand the mechanisms that regulate SR-BI-mediated delivery of cholesteryl ester (CE) from HDL to the liver for excretion. This proposal consists of two primary objectives that will test the overall hypothesis that cholesterol flux via RCT is dependent on the engagement of functional HDL particles with an oligomeric assembly of highly- structured SR-BI. Aim 1 is designed to determine the oligomeric organization of the transmembrane (TM) domains of SR-BI and their role in facilitating the selective uptake of HDL-CE. First, we will use cutting-edge NMR strategies to determine the high-resolution structures and dimerization properties of the TM domains of SR-BI. Next, we will design structure-guided mutants of SR-BI to define the requirement of ligand-induced changes in proximity and orientation of the TM domains of SR-BI in the selective uptake of HDL-CE. Together, these studies will test the hypothesis that the structural organization and conformational flexibility of the TM domains of SR-BI are critical in mediating the selective uptake of HDL-CE into the plasma membrane. In Aim 2, we will use a series of cell-based assays and in vivo experiments to determine the downstream consequences of an impaired HDL/SR-BI interaction. First, we will determine the mechanisms by which oxidatively-modified HDL prevents efficient selective uptake of HDL-CE. Second, we will determine whether SR-BI deficiency produces dysfunctional HDL particles in vivo. Finally, we will determine the effects of human mutants of SCARB1 (the human SR-BI gene) on RCT and atherosclerosis. This set of experiments will help us test the hypothesis that disruptive receptor/ligand interactions prevent HDL clearance via SR-BI-mediated selective uptake and RCT, and result in the formation of dysfunctional HDL particles that promote atherogenesis. Together, these studies will identify (i) the mechanisms by which HDL-induced conformational changes associated with the SR-BI oligomeric complex facilitate the selective uptake process and (ii) how impaired receptor/ligand interactions impede the selective uptake process and decrease RCT, resulting in the formation of dysfunctional, atherogenic HDL particles. We anticipate the findings from these studies will help identify novel therapeutic strategies for treating hypercholesterolemia and its associated pathologies such as atherosclerosis.